The Transient Receptor Potential Vanilloid 1 channel (TRPV1) is a key component of nociceptors and is also a mechanosensor in the urinary bladder and osmosensor in neurons. The water channel Aquaporin-4 (AQP-4) is found in glial cells in the CNS and is critical for water homeostasis in the brain. Although both TRPV1 and AQP-4 are important in normal body function and several pathological conditions, little is currently known about how the functions of these channels are modified by bilayer composition or their membrane microdomain locations. These are fundamental issues because the functions of many ion channels have been shown to be modified by specific membrane lipids such as phosphatidylinositol-4,5 bisphosphate (PIP2), as well as by membrane material properties controlled by the presence of cholesterol. Moreover, some channels are sequestered into cholesterol-rich plasma membrane microdomains called rafts. This application uses patch clamp electrophysiology and micropipette aspiration to measure the role of bilayer cholesterol concentration on the channel properties of TRPV1 and AQP-4, respectively. In addition, a variety of techniques, including confocal microscopy and freeze-fracture electron microscopy, will be employed to determine the microdomain locations of TRPV1 and AQP-4 and test possible mechanisms for the sequestering of channels into rafts. Our hypotheses are that: (1) specific protein-lipid and protein- protein interactions, including homo-oligomerization of the channels, are key factors in sorting channels between microdomains, (2) bilayer material properties (as controlled by phospholipid hydrocarbon chain composition and cholesterol content) modulate the function of both TRPV1 and AQP-4, and (3) acylated polybasic cytoplasmic proteins can sequester key regulatory lipids such as PIP2 into raft domains in the cytoplasmic leaflet of membrane bilayers.